Development of real-time PCR assay for quantitative detection of Clostridium septicum.

Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum (C. septicum) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene (csa), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non-septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum-containing samples from turkey farms.

Targeted detection and molecular epidemiology of turkey coronavirus spike gene variants in turkeys and chickens.

Turkey coronavirus (TCoV) is a member of the Avian coronavirus species with infectious bronchitis virus (IBV), which is considered to be the source of TCoV. These 2 viruses are highly similar in all regions of their genomes, except for the spike gene, which is necessary for virus attachment. Although TCoV causes severe enteric disease in turkey poults, it does not cause clinical disease in chickens. However, considering that TCoV can infect chickens, it is important to distinguish TCoV from IBV in chickens. This is particularly true for chickens that are housed near turkeys and thus might be infected with TCoV and serve as a silent source of TCoV for turkeys. We developed and validated a real-time PCR assay to detect the spike gene of TCoV and sequenced a portion of this gene to evaluate the molecular epidemiology of TCoV infections associated with a commercial turkey premises in the United States in 2020-2021. We identified natural infections of TCoV in chickens, and based on the molecular epidemiology of the viruses detected, these chickens may have served as a source of infection for the commercial turkey premises located nearby.

Comparing various euthanasia devices and methods on 8 and 12-week-old turkey hens.

On-farm euthanasia of poultry is a necessity for minimizing disease spread and removing sick or injured birds to maintain optimum animal welfare. There are numerous methods that are approved for euthanasia of poultry by organizations like the American Veterinary Medical Association; however, all approved methods are not easily carried out on-farm or as effective as one another. Therefore, the objective of this study was to compare several captive bolt devices (Turkey Euthanasia Device, Zephyr-EXL, Jarvis Stunner, Experimental Crossbow), mechanical cervical dislocation (Broomstick method [BRM] and Koechner Euthanasia Device [KED]), and manual cervical dislocation (MAN) methods on 8 and 12-week-old turkey hens. Each method was assessed for impact on loss of brain stem reflexes, euthanasia success, and torn skin. The cervical dislocation techniques were also analyzed via radiograph for proper dislocation. Furthermore, each device was assessed for physical parameters. Turkeys (n = 1,400) were euthanized on 20 sampling days, 10 sampling days for each age period. All methods resulted in euthanasia of all turkeys in this study. The captive bolt devices all resulted in immediate loss of nictitating membrane and pupillary reflex at both the ages tested. The cervical dislocation methods differed in both nictitating membrane and pupillary reflex cessation at both ages (P < 0.05). The pattern was the same at both ages with the KED device have longer latencies to cessation of both reflexes when compared to the BRM and MAN methods (P < 0.05). Cessation of movement was also generally longer in dislocation methods compared to captive bolt at both ages. However, captive bolt devices resulted in more lacerations of the skin in general. MAN was also found to result in less damage to the vertebrae and proper location of separation than the mechanical methods of dislocation. All methods resulted in effective euthanasia; however, captive bolt methods resulted in immediate loss of brain stem reflexes indicating that they maybe more humane than cervical dislocation methods.

Comprehensive Survey of the Litter Bacterial Communities in Commercial Turkey Farms.

The importance of microbiota in the health and diseases of farm animals has been well-documented for diverse animal species. However, studies on microbiotas in turkey and turkey farms are relatively limited as compared to other farm animal species. In this study, we performed a comprehensive survey of the litter microbiotas in 5 commercial turkey farms in the Northwest Arkansas (H, M, V, K, and R farms) including one farm with positive incidence of cellulitis (R farm). Altogether 246 boot swabs were used for 16S rRNA gene profiling of bacterial communities. At phylum level, 11 major bacterial phyla (≥0.01%) were recovered. At genus level, 13 major bacterial genera were found whose relative abundance were ≥2%. The microbial composition at both phylum and genus levels as well as their diversities varied across different farms, which were further affected by different flocks within the same farms and the ages of turkeys. Generally, the Firmicutes were higher in the flocks of younger birds, while the Actinobacteria and Bacteroidetes were higher in the flocks of the older birds. The Proteobacteria were highly enriched (47.97%) in K farm housing 56-day-old turkeys (K-56), but Bacteroidetes were found the highest in the flock C of M farm housing 63-day-old turkeys (M-C-63; 22.38%), followed by K-84 group (17.26%). Four core bacterial genera (Staphylococcus, Brevibacterium, Brachybacterium, and Lactobacillus) were identified in all samples except for those from R farm. In contrast, 24 core bacterial genera were found based in all cellulitis-associated samples (R farm), including Corynebacterium, an unknown genus of family Bacillaceae, Clostridium sensu stricto 1 (>97% similarity with C. septicum), and Ignatzschineria among others, suggesting their possible roles in etiopathogenesis of cellulitis in turkeys. Overall results of this study may provide valuable foundation for future studies focusing on the role of microbiota in the health and diseases of turkeys.

Investigating turkey enteric coronavirus circulating in the Southeastern United States and Arkansas during 2012 and 2013.

Periodic monitoring of poultry flocks in the United States via molecular diagnostic methods has revealed a number of potential enteric viral pathogens in continuous circulation in turkeys and chickens. Recently turkey integrators in the Southeastern United States and Arkansas experienced an outbreak of moderate to severe enteritis associated with turkey enteric coronavirus (TCoV), and numerous enteric samples collected from turkey flocks in these areas tested positive for TCoV via real-time reverse-transcriptase PCR (RRT-PCR). This report details the subsequent sequence and phylogenetic analysis of the TCoV spike glycoprotein and the comparison of outbreak-associated isolates to sequences in the public database. TCoVs investigated during the present outbreak grouped geographically based upon state of origin, and the RRT-PCR assay was a good indicator of subsequent seroconversion by TCoV-positive turkey flocks.